Cardiac Retention versus Engraftment of MSCsEffective delivery and enhanced retention of regenerative cells are fundamental to produce a meaningful therapeutic effect 31-35 because if the cells do not reach the target zone in the first place, they have no chance to exert any effect. A further fundamental issue is long‐term engraftment of the therapeutic cells. The latter may be, to some extent, evaluated in animal models 36 but not yet systematically in humans due to technical and safety limitations and label‐specific limitations such as any potential toxic effect on the cell 37 and/or excretion of the label from the therapeutic cell (那是, in most cases, time‐dependent) and may provide a false signal of the cell presence (cell vs. label presence) 38.

Engraftment rate of MSCs appears to be rather low 28. This phenomenon contradicts many preclinical and clinical observations in which robust beneficial effects of MSCs transplantation, such as a decrease in fibrosis, the stimulation of angiogenesis, and the restoration of contractile function, have been observed. Using an improved delivery technique, our group has recently achieved a high and reproducible retention rate (≈30%) of 99Tc‐labeled WJ‐MSCs in the peri‐infarct zone in humans after recent myocardial infarction 39.

Among the key MSCs mechanisms of action, paracrine secretion 40-43 and cell–cell interactions 44-46 appear to be most important. With these mechanisms, repeated administration of the therapeutic cells may be far more relevant to the therapeutic effect than the focus on long‐term engraftment.

Secretion of Diverse Compounds is a Unique Feature of MSCsMSCs secrete various cytokines, including hematopoietic cell proliferation and differentiation signals such as interleukin‐6, fms‐like tyrosine kinase 3 ligand, a granulocyte and macrophage colony‐stimulating factors 47, 48. They are able to induce cardioprotection via inhibition of cardiomyocyte apoptosis around the area of administration through secretion of anti‐apoptotic and angiogenic factors, such as secreted frizzled‐related protein 2, which modulates the Wnt signaling pathway 41, and vascular endothelial growth factor (VEGF), which stimulates angiogenesis 40. The secretion of proangiogenic molecules is crucial for neovasculogenesis in infarcted hearts, because MSCs lacking VEGF are less effective 40.

有趣的是, soluble cytokines and remodeling factors are not the only agents secreted by MCSs. Exosomes are small extracellular vesicles that may contain microRNAs and induce biological effects, even at distant locations. MSCs have been shown to secrete exosomes that decrease infarct size in a mouse model of myocardial ischemia/reperfusion injury 43.

The most remarkable feature of MSCs is their moderate HLA class I expression and their lack of HLA class II expression, thus resulting in their immunoprivilege 4. In many clinical trials MSCs have been found not to trigger immunologic reactions for as long as 12 months post‐transplantation 52. In contrast, they are known to have immunosuppressive properties, 例如, by promoting monocyte maturation toward anti‐inflammatory type M2 macrophages and producing soluble mediators such as transforming growth factor‐β1, hepatocyte growth factor, prostaglandin E2, indoleamine 2,3‐dioxygenase, heme oxygenase‐1, soluble HLA‐G5, and anti‐inflammatory interleukin 10 [53]. MSCs also arrest B cell and dendritic maturation, downregulate the activating receptors of natural killer cells, suppress proliferation of both T helper cells and cytotoxic T cells, and inhibit T cell production of pro‐inflammatory cytokines 54. Owing to their immunomodulatory properties, MSCs are used to treat graft‐versus‐host disease 55 and may resolve inflammation in infarcted hearts.

Through direct and indirect communication with cells at injured sites, MSCs recruit other stem cells to facilitate regeneration of injured tissue. One example of such interactions is the SDF‐1α/CXCR4 axis, which regulates homing of hematopoietic stem cells to the injured myocardium 56. 此外, cardiomyocytes can reenter the cell cycle after treatment with some cytokines secreted by MSCs (例如, TGFβ). These observations suggest that MSCs can trigger the repair of injured tissue. These intrinsic features of MSCs make them ideal candidates for regenerative cardiac therapy. 数字 1 sumarizes biological mechanisms of MSCs action.

Preclinical Cardiovascular Studies Involving MSCsMost of the aforementioned cellular mechanisms through which MSCs act in CVDs were originally identified in animal studies. The potential of MSCs to differentiate into cardiomyocytes and engraft into the myocardium has been shown in pioneering experiments in mice 28, which have revealed expression of desmin, β‐myosin heavy chain, α‐actin, cardiac troponin T, and phospholamban, as well as sarcomeric organization of the contractile proteins, in the left ventricles of mice injected with human BM‐MSCs. That and another animal study 57 have shown that the beneficial effects after MSCs injection exceed those attributable to simple differentiation and engraftment of MSCs. Owing to their immunosuppressive properties, MSCs have been found to ameliorate conditions related to non‐ischemic cardiac disorders by resolving inflammation and improving cardiac function via paracrine actions in a rat model of acute myocarditis 58.

The percutaneous injection of allogeneic MSCs into infarcted swine hearts has been found to result in long‐term engraftment, improvement in the ejection fraction, decreased scar tissue formation, and benefits to general cardiac function. 此外, this procedure has been found to be safe and to produce immunoprivilege effects in transplanted cells, because they are not rejected by allogeneic recipients 59. The beneficial effects of MSCs are not restricted to animal models of acute and/or subacute myocardial infarction. Promising results have also been observed in a chronic model of ischemic heart disease in dogs in which MSCs have been found to be able to differentiate into smooth muscle cells and endothelial cells, thus causing increased vascularity and improving cardiac function 60. Autologous MSCs have also been safely delivered into a chronic model of ischemia–reperfusion‐induced cardiomyopathy in pigs, thus resulting in structural and functional reverse remodeling 61.

Large‐animal models such as pigs are best for bridging the gap between basic research and clinical application because their size, anatomy and physiology are similar to those of humans. These models aid in not only selecting the optimal number of transplanted cells and time of transplantation but also establishing the best method (transendocardial vs. intracoronary vs. intravenous) for delivering and imaging transplanted MSCs 62. Although results in preclinical studies are very promising, showing improvement in a wide range of cardiac functions—increased ejection fraction, decrease in scar tissue, reversed remodeling, improved contractility, augmented heart perfusion, and increased blood vessel density 28-30, 40, 43, 53, 58, 61, 63, 64—the long‐term assessment of the safety and efficacy of MSCs is still needed.

Effect of MSCs Transplantation in Acute Myocardial InfarctionIn trials focused on the application of MSCs in acute/subacute myocardial infarction, BM‐MSCs have commonly been used (表 1). In one pioneering study, the short‐term (6 个月) safety of intravenous injections of allogeneic MSCs has been analyzed. No arrhythmogenicity or tumorigenicity was observed, and global symptom scores and ejection fractions tended to improve versus the effects in the placebo group 67. 在另一项研究中, BM‐MSCs have been found to be safe for small group of patients with acute myocardial infarction during a 5‐year follow‐up 68.

WJ is also a promising source of MSCs for clinical application in treating acute myocardial infarction. WJ‐MSCs have been shown to be safe and beneficial in two independent studies 13, 70, and had also positive effects on infarct size and left ventricular contractility 59.

In a study using a different design—CADUCEUS—the application of cardiospheres (mixture of autologous MSCs with CSCs) has been performed. This study has found a moderate decrease in scar tissue and increased viable heart mass and contractility in the treatment group; 然而, there was no change in ejection fraction 71. One‐year follow‐up showed that the safety and therapeutic effects of the intervention were maintained 72.

Efficiency of MSC Transplantation Is Highest in Chronic Ischemic CardiomyopathyChronic ischemic cardiomyopathy is another cardiovascular disorder in which MSCs are being intensively evaluated and are thought to be highly efficient (表 1). In the POSEIDON trial, allogeneic and autologous transendocardial applications of BM‐MSCs have been compared. Both types of cells delivered similar effects—improvement in ejection fraction and a decrease in scar size within 1 year after intervention 73.

In the TAC‐HFT trial, the effects of BM‐MSCs and bone marrow mononuclear cells (BMMNCs) have been compared. Neither cell type triggered serious adverse effects; 然而, 的BM-MSC, but not BMMNCs, caused a decrease in infarct size and improvements in contractility and overall quality of life; 然而, no changes in ejection fraction have been observed 74.

的BM-MSC’ beneficial effects in treating chronic ischemic cardiomyopathy are clear, but the effects tend to be limited and localized to the injection site. In the PROMETHEUS study, patients undergoing coronary artery bypass grafting received autologous MSCs. An 18‐month follow‐up showed improved contraction and perfusion and decreased scar tissue size in injected segments. -, the small number of participants and the lack of a placebo group restricts the degree to which these results can be generalized 75.

The effects of MSCs transplantation may be limited not only by the site of injection but also by the number of transplanted cells. Most of the aforementioned trials used dose‐escalation approaches (ranging from 12.5 × 106 - 11 × 108), whereas the ongoing TRIDENT trial—a phase II clinical trial (NCT02013674)—intends to establish the optimal number of transendocardially transplanted allogeneic MSCs, which should at least correspond to the number of cells lost during myocardial infarction while still being a number that is possible to culture and inject.

Bone marrow is not the only source of MSCs that has been tested for treating chronic ischemic cardiomyopathy. AT‐MSCs also yield improvements in total left ventricular mass, heart contractility and perfusion in no‐option patients with chronic ischemic cardiomyopathy, as shown by the PRECISE study 76. An ongoing phase II trial (CONCERT‐CHF) is testing the safety and efficacy of transendocardial injections of autologous MSCs together with c‐kit‐positive CSCs in patients with chronic heart failure.

A slightly different approach involves pretreatment of MSCs with cytokines before transplantation. In the C‐CURE study, MSCs were preconditioned with a cardiogenic cytokine cocktail before application. Increase in the ejection fraction, end‐systolic volume, 6‐minute walking distance and general quality of life were observed, with no systemic toxicity or adverse effects within 2 年份 77. In this approach, MSCs with an increased commitment to a cardiopoietic lineage are believed to be more promising than unstimulated MSCs. The C‐CURE results inspired the multinational CHART‐1 trial, conducted in 39 医院. A recent update from this study has demonstrated the safety of cardiogenic conditioned BM‐MSCs from patients 39 weeks after transplantation 78.

A similar approach has been used in the MyStromalCell study, in which patients received VEGF‐stimulated AT‐MSCs 79. In that study, prior to transplantation, AT‐MSCs were stimulated to differentiate toward an endothelial lineage by culturing for 7 days in VEGF‐A165‐stimulation medium.

有趣的是, there are no current trials making direct comparisons of the effects of MSCs from different sources (例如, AT‐MSCs vs. 的BM-MSC) in the treatment of any cardiac disorder. 同样, no studies have compared cell delivery methods in this manner. This lack of information complicates making assumptions about optimal cell sources or delivery methods.

Studies to date have generally provided optimistic observations concerning the application of MSCs in the treatment of cardiovascular disorders (acute or chronic).

In several models, MSCs have been shown to decrease scar tissue size, increase perfusion and contractility of the injured heart, induce neovasculogenesis and antifibrotic effects in damaged cardiac tissue, and generally improve quality of life. -, there is still a need for large, comprehensive, randomized controlled multicenter studies comparing crucial features of MSCs application in CVDs (例如, source and number of cells, culture conditions, 时间, and method of application). Several such studies are in progress, thus warranting cautious optimism with regard to the clinical application MSCs in the near future. 表 1 presents selected clinical trials involving MSCs in cardiovascular disorders.

Enhancing the Efficiency of MSC Therapy: Future GoalsDespite the promising results of clinical studies involving MSCs, constant efforts to enhance MSC performance are being made, primarily because effects observed in preclinical studies are stronger than those in clinical trials. To achieve the best clinical results, optimal conditions for transplantation must be established. These conditions involve duration of the disease (acute or chronic disorder); the dose of cells applied; the overall patient condition, sex and age of the patient; and the age of the cell donor in cases of allogeneic transplants.

The method of cell delivery (intracoronary vs. transendocardial vs. intravenous) is also being debated 37. On the basis of the conclusions of cardiovascular clinical trials, the transendocardial application of 20–100 × 106 MSCs in treating chronic ischemic cardiomyopathy may deliver the best results. -, there is a lack of comprehensive studies discussing these issues and showing a reliable efficacy of MSCs transplantation that exceeds the efficacy of standard procedures alone. Ultimately, combined therapies may prove most viable. An interesting concept is to test MSCs as an adjunctive therapy in patients receiving left ventricular assist devices 80.

There is also a lack of data showing the optimal source of MSCs for transplantation. As shown in basic science studies, MSCs can differ across sources in their regenerative potential, 那是, in their level of secreted trophic factors or propensity toward different lineages. -, there are many discrepancies among published data regarding the properties of BM‐, AT‐, and WJ‐MSCs. 因此, comprehensive studies are needed to obtain consistent results. Such studies may also improve cell preparation methods for specific clinical trials.

The absence of differences between the effect of autologous and allogeneic BM‐MSCs used in clinical studies for the treatment of ischemic cardiomyopathy has previously been reported 73. -, allogeneic cells have advantages over autologous cells in that they can be prepared, expanded and characterized more quickly as off‐the‐shelf‐products that are ready to be applied when needed. Our 13 and another group 70 have suggested the use of an innovative source of allogeneic MSCs, WJ, for treating cardiac disorders. In the CIRCULATE study, WJ‐MSCs will be isolated from umbilical cords and characterized on the basis of their molecular features and their ability to treat cardiac disorders both in in vivo models and in a clinical trial. This approach may address the unmet needs regarding the clinical application of MSCs, which include but are not restricted to the poor availability of abundant autologous cells in the short time period after heart failure. It has been estimated that myocardial infarction is associated with loss of approximately 109 cardiac myocytes 65. 从而, that is the order of magnitude of cells necessary for transplantation within days after a cardiac incident. An off‐the‐shelf approach appears to be more feasible than autologous cell expansion to meet this need.

此外, because cardiovascular disorders mainly affect elderly people with comorbidities (例如, 糖尿病), it is safe to assume that their autologous cells would also suffer “comorbidities,” thereby diminishing the long‐term therapeutic effects of transplanted autologous cells. This risk is overcome via the application of “healthy, young” allogeneic cells.

Despite numerous clinical studies showing beneficial effects of MSCs in treating cardiovascular disorders, some authors have called into question the nature of MSCs, and have even suggested that MSCs and fibroblasts cannot be distinguished on the basis of morphology, cell‐surface markers, differentiation potential or immunologic properties 85-87. This highlights the importance of defining MSCs properly and may reflect the trap of inaccurate nomenclature, because no stem‐cell nature would be expected in fibroblasts.

有趣的是, despite of all abovementioned concerns, the level of improvement in left ventricular ejection fraction observed in cell therapy trials is comparable to the levels observed with the use of the most effective pharmacological treatments 39. One common criticism of cellular therapies to stimulate myocardial repair and regeneration involves their seemingly small effect on myocardial contractility, typically evaluated as the left ventricular ejection fraction (LVEF). It needs to be noted that typically reported improvements in LVEF in patients with heart failure by ≈2%–4% 88 are not different from the typical effect of widely recognized pharmacological therapies (例如, beta‐blockers +2.9% 89, angiotensin receptor blockade +1.3% 90, aldosterone inhibition +2.0% 91 or cardiac resynchronization therapy +2.7% 92. It is expected that improvements in cell therapy including the use of unlimited cell sources, reproducible cell harvest, preparation protocols and standardized delivery methods taking advantage of the latest technology will translate into advancing beyond the magnitude of the effect of contemporary pharmacotherapy.

结论A number of unique features of MSCs discussed above make them unique and promising therapeutic agents, in the field of stem cell research. Rather than being typical stem cells that differentiate into effector cells, which directly trigger the regeneration of damaged tissues (similar to construction workers at a construction site), they act as governing cells that secrete mediators and/or directly interact with other cells and subsequently stimulate or recruit those cells to perform regenerative actions (similarly to construction site supervisors). To conclusively demonstrate these effects, additional well‐designed randomized multicenter studies are needed before MSCs treatment can become a therapy of choice for the fundamental health problem worldwide, 心血管病. Allogeneic MSCs are particularly interesting as therapeutic agents because they are not only free of fundamental biologic limitations of autologous cells 93 but also can be used as “off‐the‐shelf” therapeutic agents 13.

In a mutual relationship to clinical trials, important issues that need to be addressed at the pre‐clinical and early clinical stage of MSCs applications involve (一个) reduction or elimination of cell antigenicity to reduce or eliminate rejection 54, (b) continued development of improved delivery techniques to enhance myocardial retention and engraftment 62, 和 (C) cell engineering and /or preconditioning 94 to enhance regenerative capacities and enhance survival. Recent study in subacute myocardial infarction in humans indicates an unprecedented high‐grade (systematically 30%–35%) transcoronary施用天然低免疫原性的WJ-MSC的心肌摄取 39. 这超过了≈5倍其它细胞类型的心肌摄取 (如非选择或所选择的骨髓造血或间充质细胞) 95 亚急性人类心肌梗死, 说明一个重要的临床研究方向 96.